Zinc is an essential micronutrient for plants, but it is toxic in excess concentrations. In Arabidopsis, additional iron (Fe) can increase Zn tolerance. We isolated a mutant,zinc tolerance induced by iron 1, designatedzir1, with a defect in Fe-mediated Zn tolerance. Using map-based cloning and genetic complementation, we identified thatzir1has a mutation of glutamate to lysine at position 385 on γ-glutamylcysteine synthetase (GSH1), the enzyme involved in glutathione biosynthesis. Thezir1mutant contains only 15% of the wild-type glutathione level. Blocking glutathione biosynthesis in wild-type plants by a specific inhibitor of GSH1, buthionine sulfoximine, resulted in loss of Fe-mediated Zn tolerance, which provides further evidence that glutathione plays an essential role in Fe-mediated Zn tolerance. Two glutathione-deficient mutant alleles ofGSH1,pad2-1andcad2-1, which contain 22% and 39%, respectively, of the wild-type glutathione level, revealed that a minimal glutathione level between 22 and 39% of the wild-type level is required for Fe-mediated Zn tolerance. Under excess Zn and Fe, the recovery of shoot Fe contents inpad2-1andcad2-1was lower than that of the wild type. However, the phytochelatin-deficient mutantcad1-3showed normal Fe-mediated Zn tolerance. These results indicate a specific role of glutathione in Fe-mediated Zn tolerance. The induced accumulation of glutathione in response to excess Zn and Fe suggests that glutathione plays a specific role in Fe-mediated Zn tolerance in Arabidopsis. We conclude that glutathione is required for the cross-homeostasis between Zn and Fe in Arabidopsis.